1. Field of the Invention
The present invention relates to a method for separating viruses from a contaminant-containing liquid medium, wherein the viruses are first adsorbed to a first adsorbent having cationic groups and subsequently desorbed therefrom and wherein subsequently a liquid medium containing the viruses and contaminants is contacted with a second adsorbent having cationic groups in order to adsorb the contaminants.
2. Description of the Related Art
Efficient methods for separating viruses from biotechnological liquids, which frequently contain undesired contaminants, are becoming increasingly important in medicine and biotechnology.
Viruses, virions or viral particles consist of a nucleic acid (deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) and a protein coat, also termed a capsid. Some viruses are additionally surrounded by a membrane, which is referred to as a viral envelope, or have other additional constituents. Such viruses which have a viral envelope in addition to the capsid are termed enveloped, and viruses without such an envelope are referred to as nonenveloped. Viroids have neither a capsid nor a viral envelope. A virus itself is not capable of metabolic processes, and it therefore requires host cells for propagation. Viruses attack the cells of eukaryotes (plants, fungi, animals) and prokaryotes (bacteria and archaea). Viruses which utilize prokaryotes as hosts are termed bacteriophages.
The influenza virus is an enveloped virus particle having a diameter of from 80 to 120 nm, in the envelope of which differing numbers of proteins and glycoproteins are embedded. The genome of almost all influenza viruses consists of eight negative-sense RNA segments. The main proteins of the envelope are hemagglutinin (HA) and neuraminidase (NA). Viral mutations, particularly with respect to possible alterations to hemagglutinin, can considerably increase the risk of infection for the potential host.
Virus-like particles (VLPs) are virus particles which are produced by biotechnological means. They do not contain any viral nucleic acids and are therefore not capable of multiplying in the target cells. VLPs are required in virology and in immunology for studying viruses and cellular functions. These particles are not empty, since they would otherwise become unstable; instead they are packed with either, nonspecifically, nucleic acids or with nonfunctional DNA or RNA. In addition, proteins can be packed in a specific manner.
Natural and/or recombinant viruses can be used as vaccines in medicine. Vaccines are preventative or therapeutic agents which bring about their effect by stimulating the immune system of an individual. Live attenuated vaccines contain weakened viruses which can still multiply and induce an immune response, i.e., are immunogenic, but do not generally cause a disease, i.e., are not pathogenic. Dead vaccines contain inactivated or killed viruses or constituents of viruses.
Another way of using viruses in medicine is to be found in gene therapy. Gene therapy refers to the insertion of genes into the cells of an individual for the treatment of inherited diseases or gene defects. The introduction of said genes can compensate for a gene defect. A gene defect is present when a living being lacks a gene or has a mutation which results in the gene product (e.g., a protein) not being formed or not being able to carry out its function properly. During gene therapy, cells are removed from the body. Said cells receive the new (therapeutic) gene and are subsequently reintroduced into the body (ex vivo). The use of viral vectors makes it possible to carry out gene therapy directly in the body (in vivo). Here, retroviruses or adenoviruses are used in most cases for transferring DNA segments into the somatic cells of the patient.
The production of viruses which are used either as vaccines or as vectors in gene therapy is of increasing relevance to biotechnology. Production is carried out in several steps:    1. generating a pathogen/antigen,    2. producing the antigen in an appropriate system (e.g., cell culture, chick embryos),    3. purifying the antigen and    4. formulating the vaccine or the vector by addition of auxiliary agents, adjuvants, stabilizers, preservatives, etc.
After culturing viruses in cell cultures (e.g., MRC-5, vero, PER.C6) or in chicken eggs, it is necessary to separate the viruses from the contaminants (e.g., host cell proteins, DNA or endotoxins) in order to obtain them in a pure form for the desired application. Moreover, it is advantageous to separate infectious molecules or particles from noninfectious ones. Viral properties, such as the isoelectric point (pI), surface hydrophobicity, presence of an envelope and the hydrodynamic diameter, can be used for the purposes of the purification. Purification methods based on the size of the viruses are known in the prior art. They include, for example,                density-gradient ultracentrifugation (in a CsCl or sucrose gradient; the high capital costs are a disadvantage),        ultrafiltration and microfiltration using planar or hollow-fiber membranes,        precipitation (e.g., using polyethylene glycol or ammonium sulfate) or        size-exclusion chromatography using chromatography gels, for example based on agarose. The disadvantages of the last-mentioned method are low flow rates and the low process speed.        
For the degradation of host-cell DNA, an enzyme, for example the endonuclease BENZONASE®, is often used. The disadvantage of this method is the very high enzyme costs. Depth filters are used to remove cells and/or cell fragments when purifying viruses.
The adsorption of viruses to solid phases as a result of chromatographic purification is of great significance in virus purification, especially on a process scale. Adsorbents are porous solids which can bind selectively to particular components of fluids via functional surface groups referred to as ligands. Target substance(s) and/or contaminant(s) are referred to as adsorbands, and they can also be several different substances. Adsorbands can be individual molecules, associate or particles and are preferably viruses, viral constituents, virus-like particles, proteins or other substances of biological origin.
The binding of the adsorbands to the adsorbent can be reversible or irreversible, and in any case it allows the separation thereof from the fluids, which are generally aqueous liquids and are termed media hereinafter. The term “elution” covers the desorption of an adsorband from the adsorbent and the associated wash steps, etc. The liquid used for the elution is the eluent. The components can be one or more target substances and/or one or more contaminants. Target substances are valuable substances which are to be obtained in enriched or pure form from the medium. Target substances can, for example, be viruses. Contaminants are substances whose absence or removal from the fluid is necessary or desirable for technical, regulatory or other reasons. Contaminants can, for example, be host-cell proteins, amino acids, nucleic acids, endotoxins, protein aggregates, ligands or parts thereof. For the removal of contaminants, which is referred to as “negative adsorption” (also termed “flow-through” (FT)), the adsorption can/must proceed irreversibly if the adsorbent is to be used only once. In the case of the adsorption of target substance(s), the process has to proceed reversibly (also termed “bind-and-elute” (B&E)). Either a mere enrichment or a separation into several target substances can be carried out, and in the latter case either the adsorption, the desorption or both can be done selectively.
Conventional adsorbents for chromatography are particulate in form and are operated in the form of packings in columns. Since viruses are typically up to 1000 nm in size, conventional chromatography gels having pore sizes in the range of 30-400 nm are usually unsuitable for virus purification. The viruses can only bind to the outer surface of the particles, and as a result only low binding capacities are achieved. Various ligands have already been used in virus purification: anion exchangers (AEX), cation exchangers (CEX), affinity ligands (AF), ligands for hydrophobic interaction chromatography (HIC) or complexing ligands for immobilized metal ion affinity chromatography (IMAC).
In the prior art, numerous methods for separating viruses from biotechnological fluids by means of various chromatography matrices having ion-exchanging ligands have been described.
WO 03/078592 A2 describes a method for purifying adenoviruses, obtained from cell lysates, by means of two anion-exchanger filters. The adenovirus is first bound reversibly in “bind-and-elute” mode on a first anion-exchange filter. Thereafter, the eluate obtained is bound reversibly in “bind-and-elute” mode on a second anion-exchange filter following nuclease treatment to degrade nucleic acid contaminants.
U.S. Pat. No. 6,261,823 B1 describes a method for purifying adenoviruses by means of a first anion-exchanger chromatography step using DEAE Fractogel followed by a second size-exclusion chromatography step using the gel Superdex 200. The virus is adsorbed reversibly in “bind-and-elute” mode on the anion exchanger and subsequently eluted in the first step.
U.S. Pat. No. 5,837,520 describes a method for purifying viral vectors obtained from cell lysates following treatment with nucleic acid-cleaving enzymes. In a first step, the viral particles are treated by means of a first cation- or anion-exchanging chromatography gel and, in a second step, they are treated with an affinity chromatography gel on which metal ions capable of chelating are immobilized. Alternatively, in the second step, a chromatography step based on hydrophobic interactions can be carried out.
U.S. Pat. No. 6,008,036 describes a method for virus purification by means of two ion exchangers in two steps, with an anion-exchanging chromatography matrix being used for the second step when a cation-exchanging matrix is used in the first step, and vice versa.
EP 1 878 791 A1 describes the purification of influenza viruses by means of various anion exchangers, including gels, monoliths and membrane adsorbers. The described method provides only low yields in the region of not more than 50% for influenza viruses.
B. Kalbfuss et al. disclose in “Journal of Membrane Science”, 299 (2007), 251-260, the purification of influenza A viruses from cell cultures by means of Sartobind® D MA75 and Sartobind® Q anion-exchange membranes. The virus is bound reversibly to this strong or weak anion exchanger and subsequently eluted. To increase the selectivity of the respective membrane for viruses, the authors propose placing upstream of the anion-exchange step a pretreatment step in which the accompanying contaminants consisting of nucleic acids are separated from the influenza viruses.
B. Kalbfuss (University of Magdeburg dissertation (2009): “Downstream Processing of Influenza Whole-Virions for Vaccine Production”) reports on the binding of influenza viruses to a Sartobind® Q membrane adsorber, which is a strong anion exchanger, in comparison with the binding of said viruses to a Sartobind® D membrane adsorber, which is a weak anion exchanger. The virus yields when using the Sartobind® Q membrane adsorber are, at 86%, greater than the virus yields which are achieved when using the Sartobind® D membrane adsorber (38%). The low isoelectric point of the accompanying contaminants (DNA) allows the strong adsorption thereof. By adjusting the pH and ionic strength, DNA can be adsorbed, with the viruses being conducted through the anion exchanger without being adsorbed. In experiments to separate DNA as contaminants from influenza viruses, it was found that very high salt concentrations are required in order to suppress the virus adsorption. At a concentration of 0.15 M NaCl, the virus adsorbs completely to the membrane adsorber, and, at a concentration of 0.7 M NaCl, 10% of the virus was adsorbed to Sepharose® Q XL. However, the high salt concentration leads to the breakthrough of DNA.
The convectively permeable chromatographic materials known in the prior art, such as membrane adsorbers (e.g., Sartobind® product line from Sartorius Stedim Biotech GmbH) or monoliths from BIA Separations, were originally developed and optimized for protein purification, the aim being high binding capacities for proteins. When purifying viruses, the intention is to remove proteins (e.g., host-cell proteins or endotoxins) or nucleic acids, as contaminants, from the virus, as target product. When using the aforementioned methods known in the prior art using ion exchangers, not only the viruses but also the aforementioned contaminants are adsorbed, and this leads to low purities and low virus yields following the desorption of the virus from the membrane adsorber.
T. Vicente et al. describe in “Gene Therapy” 2009, 1-10, a three-stage method for purifying baculoviruses, comprising an opening step of depth filtration, a second step of ultrafiltration or diafiltration and a final step of purification of baculoviruses by means of reversible binding to a Sartobind® D MA15 anion-exchanger membrane adsorber.
It is an object of the present invention to provide a method which overcomes the aforementioned disadvantages of the prior art (low purity of the viruses owing to the presence of contaminants and low yields of viruses) and which makes it possible to provide viruses from biotechnological fluids in high yields and high purity in a rapid and cost-saving manner.